248 L C Hernández- CABLES1 mutations in 24:8 379–392 Research Ramírez et al. Cushing’s disease Open Access Loss-of-function mutations in the CABLES1 gene are a novel cause of Cushing’s disease Laura C Hernández-Ramírez1, Ryhem Gam2, Nuria Valdés1,3, Maya B Lodish1, Nathan Pankratz4, Aurelio Balsalobre2, Yves Gauthier2, Fabio R Faucz1, Giampaolo Trivellin1, Prashant Chittiboina5, John Lane4, Denise M Kay6 Aggeliki Dimopoulos7, Stephan Gaillard8,9, Mario Neou8, Jérôme Bertherat8,10, Guillaume Assié8,10, Chiara Villa8,11,12, James L Mills7,*, Jacques Drouin2,*, and Constantine A Stratakis1,* 1Section on Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, Maryland, USA 2Laboratoire de Génétique Moléculaire, Institut de Recherches Cliniques de Montréal (IRCM), Montréal, Québec, Canada 3Service of Endocrinology and Nutrition, Hospital Universitario Central de Asturias, Instituto Universita- rio de Oncología del Principado de Asturias, Universidad de Oviedo, Oviedo, Spain 4Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, Minnesota, USA 5Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH), Bethesda, Maryland, USA 6Newborn Screening Program, Wadsworth Center, New York State Department of Health, Albany, New York, USA 7Division of Intramural Population Health Research, Epidemiology Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, Maryland, USA 8 Endocrine-Related Cancer Endocrine-Related Institut Cochin, INSERM U1016, CNRS UMR8104, Université Paris Descartes, Paris, France 9Department of Neurosurgery, Hôpital Foch, Suresnes, France 10 Service d’Endocrinologie, Cochin Hospital, Assistance Publique Hôpitaux de Paris, Paris, France Correspondence 11 Department of Pathological Cytology and Anatomy, Hôpital Foch, Suresnes, France should be addressed 12 Department of Endocrinology, CHU de Liège, University of Liège, Liège, Belgium to C A Stratakis *(J L Mills, J Drouin and C A Stratakis contributed equally to this work) Email [email protected] Abstract The CABLES1 cell cycle regulator participates in the adrenal–pituitary negative feedback, Key Words and its expression is reduced in corticotropinomas, pituitary tumors with a largely f Cushing’s disease unexplained genetic basis. We investigated the presence of CABLES1 mutations/copy f corticotropinoma number variations (CNVs) and their associated clinical, histopathological and molecular f whole-exome sequencing features in patients with Cushing’s disease (CD). Samples from 146 pediatric (118 f germline mutation germline DNA only/28 germline and tumor DNA) and 35 adult (tumor DNA) CD patients were screened for CABLES1 mutations. CNVs were assessed in 116 pediatric CD patients (87 germline DNA only/29 germline and tumor DNA). Four potentially pathogenic missense variants in CABLES1 were identified, two in young adults (c.532G > A, p.E178K and c.718C > T, p.L240F) and two in children (c.935G > A, p.G312D and c.1388A > G, and p.D463G) with CD; no CNVs were found. The four variants affected residues within or close to the predicted cyclin-dependent kinase-3 (CDK3)-binding region of the CABLES1 protein and impaired its ability to block cell growth in a mouse corticotropinoma cell http://erc.endocrinology-journals.org © 2017 The authors This work is licensed under a Creative Commons DOI: 10.1530/ERC-17-0131 Published by Bioscientifica Ltd. Attribution 3.0 Unported License. Printed in Great Britain Downloaded from Bioscientifica.com at 09/24/2021 06:28:58PM via free access 10.1530/ERC-17-0131 Research L C Hernández- CABLES1 mutations in 24:8 380 Ramírez et al. Cushing’s disease line (AtT20/D16v-F2). The four patients had macroadenomas. We provide evidence for a role of CABLES1 as a novel pituitary tumor-predisposing gene. Its function might link two of the main molecular mechanisms altered in corticotropinomas: the cyclin- dependent kinase/cyclin group of cell cycle regulators and the epidermal growth factor receptor signaling pathway. Further studies are needed to assess the prevalence of CABLES1 mutations among patients with other types of pituitary adenomas and to Endocrine-Related Cancer elucidate the pituitary-specific functions of this gene. (2017) 24, 379–392 Introduction Materials and methods In recent years, germline defects in multiple genes have Pediatric Cushing’s disease cohort been linked to the pathogenesis of pituitary adenomas, We studied 146 pediatric ( 18 years at diagnosis) patients both with syndromic and isolated presentation (Caimari & < with CD who are part of a large cohort evaluated at the Korbonits 2016). In contrast with other types of pituitary outpatient clinic and/or admitted for clinical work-up tumors, corticotropinomas are infrequent in patients with and treatment at the National Institutes of Health (NIH) ‘classic’ multiple endocrine neoplasia and have rarely Clinical Center between 1997 and 2017 and recruited been described in the setting of familial isolated pituitary under the research protocol 97-CH-0076 (ClinicalTrials. adenoma (FIPA) (Stratakis et al. 2010, Cazabat et al. 2012). gov: NCT00001595). The Eunice Kennedy Shriver National Consequently, the germline abnormalities leading to Institute of Child Health and Human Development corticotroph cell tumorigenesis remain largely unknown. Institutional Review Board approved this study, and On the other hand, somatic mutations in the USP8 gene informed assent/consent was obtained from all the hotspot are highly prevalent among corticotropinomas patients and their parents/guardians. Clinical data were in adult and pediatric patients, but they have not been obtained directly from the patients and/or from the detected in other types of pituitary adenomas (Pérez- Clinical Research Information System. Parents and siblings Rivas et al. 2015, Reincke et al. 2015). Therefore, it might of the patients were also recruited, when available. be that the majority of the corticotropinomas are caused For all the individuals, DNA was extracted either from Endocrine-Related Cancer Endocrine-Related by disruptions in molecular pathways that are not shared a peripheral blood sample using the Maxwell 16 Blood by other pituitary tumor types. DNA Purification Kit in a Maxwell 16 Instrument (Promega The CABLES1 (Cdk5 and ABL enzyme substrate AS1015 and AS3050) or from saliva using the Oragene-Dx 1) gene (18q11.2) is a negative regulator of cell cycle collection kit and the PrepIT-L2P DNA extraction kit progression that is activated in corticotroph cells in (DNA Genotek OGD-500 and PT-L2P-45), according to response to glucocorticoids (Roussel-Gervais et al. the manufacturer’s protocols. When available, stained 2016). The physiological negative feedback exerted by histopathological sections from the corticotropinomas glucocorticoids on the corticotroph cells is often impaired were retrieved from the Department of Pathology. in corticotropinomas, and, concordantly, CABLES1 After either manual delimitation of the tumor area or protein expression is lost in around half of such tumors microdissection, DNA was extracted from unstained (Roussel-Gervais et al. 2010, Roussel-Gervais et al. 2016). sections using the Pinpoint Slide DNA Isolation System CABLES1 gene inactivation by allelic loss, aberrant splicing (Zymo Research D3001). Screening for germline mutations or promoter hypermethylation has been observed in in AIP, CDKN1B, CDKN2C, MEN1, and PRKAR1A in 74 different types of human cancers but, to our knowledge, of these patients, and GPR101 in 34 of them, as well as it has not been explored in pituitary adenomas before somatic GPR101 defects in 23 corticotropinoma DNA (Tan et al. 2003, Zhang et al. 2005, Sakamoto et al. 2008). samples from these patients has been reported before Moreover, there are no human phenotypes reported in (Stratakis et al. 2010, Trivellin et al. 2016). association with CABLES1 germline mutations (http:// Germline DNA samples from 98 patients and tumor omim.org/entry/609194, accessed: 28-03-17). Therefore, DNA samples from 28 of them were submitted for we investigated the presence of CABLES1 gene mutations whole-exome sequencing (WES) at the University of and copy number variations (CNV) in a large group of Minnesota Genomics Center. Targeted capture libraries patients with Cushing’s disease (CD). http://erc.endocrinology-journals.org © 2017 The authors Published by Bioscientifica Ltd. DOI: 10.1530/ERC-17-0131 Printed in Great Britain Downloaded from Bioscientifica.com at 09/24/2021 06:28:58PM via free access Research L C Hernández- CABLES1 mutations in 24:8 381 Ramírez et al. Cushing’s disease were generated using the Agilent QXT v5 + UTRs kit were used for missense variants and five (Splice Site for both germline and tumor samples. The germline Finder, MaxEnt, NNSplice, GeneSplicer and Human Site samples were sequenced on an Illumina HiSeq 2000 Finder) were used for splicing variants. Variants were platform producing 100 bp paired-end reads, while considered probably damaging or affecting splicing when the tumor samples were sequenced on a HiSeq 2500 the majority of the algorithms agreed; otherwise, they platform producing 125 bp paired-end reads. FASTQ were considered variants of uncertain significance (VUS). files were processed using the steps delineated in the Sanger sequencing